This proposed MIRA project employs a range of multi-disciplinary approaches toward the discovery and analysis of natural products and the biosynthetic pathways that assemble and modify complex metabolites. The proposal covers three areas that have been supported by NIGMS during the past 20 years. Each has been articulated as a Grand Challenge designed to complement our accomplishments and continue to push forward vigorously to discover new knowledge and offer solutions with high potential for improving human health. Grand Challenge I of this MIRA application is based on the exciting momentum of a highly productive and collaborative program lead by my group that focuses on the pikromycin (Pik), erythromycin (DEBS), tylosin (Tyl), curacin (Cur) and bryostatin (Bry) pathways whose detailed analysis has been further developed during the previous cycle of support. These systems each bear fascinating biochemical features that will expand our understanding of substrate selectivity, and structural characteristics that enable functional activity within and between native and engineered polyketide synthase/non-ribosomal peptide synthetase modules. Grand Challenge II of this proposal focuses on studies relating to natural product pathway tailoring enzymes. A fundamental aspect of structural diversification in secondary metabolism involves oxidative processes that contribute significantly to biological activity. This can be readily appreciated in a number of important molecules that are clinical therapeutic agents, or show significant potential as drug leads. Based on the important successes in our research relating to P450 substrate and enzyme engineering over the past four years, we have been emboldened to expand our work in exciting new directions. This includes plans to investigate a range of P450 monoxygenases that catalyze iterative oxidative processes. We will also investigate monooxygenases that catalyze C-C coupling involving substrates in both inter- and intramolecular oxidation reactions, including aromatic, alkyl and alkenyl functional groups. One of the most underexplored, yet very important classes of tailoring enzyme includes the acyl/peptidyl carrier protein dependent monooxygenases, and we propose to explore mechanisms of selectivity and proceed with efforts to expand their substrate recognition and biocatalytic properties. Grand Challenge III focuses on natural product discovery and pathway engineering. We have established the technologies and bioinformatics capabilities to readily assemble and mine genomic, and metagenomic datasets from diverse microbiome populations toward natural product gene cluster discovery, which is now poised for heterologous expression in amenable microbial hosts. The next wave of progress will rely on ready identification of the most novel pathways, and our ability to express them using facile synthetic biology methods. We plan to attack these problems with utmost energy and determination to gain access to important compounds with valuable medicinal properties.